The invention relates to diagnosis and treatment of vascular injury.
Atherosclerosis and its subsequent complications, such as myocardial infarction, stroke, and peripheral vascular diseases, are the major causes of death in developed countries. Vascular endothelial and smooth muscle cells have important roles in the regulation of normal vascular tone. Damage or dysfunction of these cells can lead to vascular diseases, such as atherosclerosis and restenosis.
Atherosclerosis is believed to be a consequence of a response of the vascular wall to injury (Ross, R., 1993, Nature 362:801-9). Upon vascular injury and various other stimuli, cytokines and growth factors from activated vascular cells promote growth and migration of vascular smooth muscle cells in a dedifferentiated status, resulting in the formation of atherosclerotic plaques.
The pathogenesis of atherosclerosis is not fully understood, and an effective therapeutic regime has not been developed to prevent or cure atherosclerosis (Ross, R., The Pathogenesis of Atherosclerosis, in Heart Disease, a textbook of cardiovascular medicine, E. Braunwald, Editor, 1992, W. B. Saunders Company: Philadelphia. pp. 1106-24; and Ross, R.: The Pathogenesis of Atherosclerosis: a Perspective for the 1990s, 1993, Nature 362:801-9). Despite extensive research, the molecular mechanisms responsible for the regulation of gene expression in vascular endothelial and smooth muscle cells are largely unknown. In particular, trans-acting factors and cis-acting elements mediating vascular cell-specific gene expression have not been identified, mainly due to the fact that only a few vascular specific genes have been identified. Furthermore, of the genes that have been characterized as endothelial cell-specific (e.g. von Willebrand factors, VEGF receptor flk-1, VCAM-1, and E-selection (Hunter, J. J., et al., 1993, Hypertension 22:608-17) or smooth muscle cell-specific (e.g., CHIP28, SM22, and gax (Gorski, D. H., et al., 1993, Mol. Cell. Biol. 13(6):3722-33), many have been found in other cell types at various levels.
The invention is based on the discovery of a novel gene the expression of which gives rise to variant isoforms, one which is specific to aortic cells, and others which are found in striated muscle cells. Accordingly, the invention features an aortic cell-specific gene, and therefore provides a substantially pure DNA (e.g., genomic DNA, cDNA or synthetic DNA) encoding an aortic-preferentially-expressed gene-1 (APEG-1) polypeptide. By xe2x80x9csubstantially pure DNAxe2x80x9d is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the APEG-1 gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
Hybridization is carried out using standard techniques such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, (1989). xe2x80x9cHigh stringencyxe2x80x9d refers to DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65xc2x0 C. at a salt concentration of approximately 0.1xc3x97SSC. xe2x80x9cLowxe2x80x9d to xe2x80x9cmoderatexe2x80x9d stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e.g. wash conditions of less than 60xc2x0 C. at a salt concentration of at least 1.0xc3x97SSC. For example, high stringency conditions may include hybridization at about 42xc2x0 C. and about 50% formamide; a first wash at about 65xc2x0 C., about 2xc3x97SSC, and 1% SDS; followed by a second wash at about 65xc2x0 C. and about 0.1% xc3x97SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to an APEG-1 gene are detected by, for example, hybridization at about 42xc2x0 C. in the absence of formamide; a first wash at about 42xc2x0 C., about 6xc3x97SSC, and about 1% SDS; and a second wash at about 50xc2x0 C., about 6xc3x97SSC, and about 1% SDS.
A substantially pure DNA having at least 50% sequence identity (preferably at least 70%, more preferably at least 80%, and most preferably at least 90%) to SEQ ID NO:1, 2, or 11, and encoding a polypeptide having a biological activity of an APEG-1 polypeptide is also within the invention. The percent sequence identity of one DNA to another is determined by standard means, e.g., by the Sequence Analysis Software Package developed by the Genetics Computer Group (University of Wisconsin Biotechnology Center, Madison, Wis.) (or an equivalent program), employing the default parameters thereof. xe2x80x9cBiological activity of an APEG-1 polypeptidexe2x80x9d is defined as the ability to inhibit the proliferation or migration of smooth muscle cells at the site of vascular injury.
The invention also includes a substantially pure DNA containing a constitutive or inducible, vascular cell-specific promoter, e.g., an APEG-1 promoter which is preferably in a vector into which an heterologous gene may be or has been cloned, and under the control of which the gene may be expressed. The promoter is preferably specific for arterial cells (e.g., cells of the aorta), and most preferably specific for vascular smooth muscle cells. DNA encoding APEG-1 may be operably linked to such regulatory sequences for expression of the APEG-1 polypeptide in vascular cells.
By xe2x80x9cpromoterxe2x80x9d is meant a minimal DNA sequence sufficient to direct transcription. Promoters may be constitutive or inducible, and may be coupled to other regulatory sequences or xe2x80x9celementsxe2x80x9d which render promoter-dependent gene expression cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 region of the native gene, or within an intron. By xe2x80x9cheterologous promoterxe2x80x9d is meant a promoter other than a naturally occurring APEG-1 promoter.
By xe2x80x9coperably linkedxe2x80x9d is meant that a coding sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
The invention also provides a method of directing vascular cell-specific expression of a protein by introducing into a vascular cell an isolated DNA containing a sequence encoding the protein operably linked to the vascular cell-specific promoter. A cell containing the DNA or vector of the invention is also within the invention.
The invention also features a substantially pure APEG-1 polypeptide (e.g., rat APEG-1 (SEQ ID NO:3) or human APEG-1 (e.g., human APEG-1 (SEQ ID NO:12)) and an antibody which specifically binds to an APEG-1 polypeptide. By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide which is separated from those components (proteins and other naturally-occurring organic molecules) which naturally accompany it. Typically, the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation. Preferably, the protein in the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, APEG-1 polypeptide. A substantially pure APEG-1 polypeptide may be obtained, for example, by extraction from a natural source (e.g., an aortic cell); by expression of a recombinant nucleic acid encoding an APEG-1 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include recombinant polypeptides derived from a eukaryote but produced in E. coli or another prokaryote, or in a eukaryote other than that from which the polypeptide was originally derived.
In another aspect, the invention provides a method of detecting injury in a sample of vascular tissue by determining the level of APEG-1 gene expression in the tissue; a decrease in the level of expression detected in the tissue sample compared to that detected in uninjured control vascular tissue indicates the presence of a vascular injury.
The invention also includes a method of inhibiting smooth muscle cell proliferation in an animal by contacting an artery of the animal with an APEG-1 polypeptide or a biologically active fragment thereof or with a compound that stimulates the APEG-1 promoter, e.g., stimulates APEG-1 expression.
In yet another aspect, the invention includes a method of making an APEG-1 polypeptide, e.g., a rat or human APEG-1 polypeptide, involving providing a cell containing DNA encoding an APEG-1 polypeptide and culturing the cell under conditions permitting expression of the APEG-1 encoding DNA, i.e., production of the recombinant APEG-1 by the cell.
The invention further features a substantially pure DNA having an APEG-1 derived enhancer sequence which regulates vascular smooth muscle cell-specific transcription of a polypeptide-encoding sequence to which it is operably linked. By xe2x80x9cenhancer sequencexe2x80x9d is meant a DNA sequence which contains one or more cis-acting elements which regulate transcription, e.g., cell specific transcription. The elements may be contiguous or separated by DNA not involved in the regulation of transcription, e.g., an enhancer element may be in a position immediately adjacent to the promoter element or up to several kilobases upstream or downstream of the transcriptional start site. The enhancer DNA is preferably derived from the 5xe2x80x2 region of a mammalian APEG-1 gene, such as that of the mouse (SEQ ID NO:17), and regulates preferential expression in vascular smooth muscle cells, e.g., aortic smooth muscle cells, of a polypeptide-encoding DNA to which it is operably linked. Preferably, the enhancer includes a sequence which hybridizes under high stringency conditions to SEQ ID NO:20 or SEQ ID NO:23, or a complement thereof. More preferably, the enhancer includes the 73 nucleotides of SEQ ID NO:20, which is located within the sequence of SEQ ID NO:17. Most preferably, the enhancer includes the 38 nucleotides of SEQ ID NO:23, which is located within the sequence of SEQ ID NO:20.
The enhancer sequence can be less than 100 nucleotides in length or less than 50 nucleotides in length.
In some embodiments, the enhancer includes less than the complete nucleotide sequence of SEQ ID NO:17, e.g., it can contain less than 2.6 kb, 2.1 kb, 1.7 kb, 1.2 kb, 700 nucleotides, 500 nucleotides, or even 100 nucleotides of SEQ ID NO:17.
In some embodiments, the enhancer contains SEQ ID NO:20 and is less than 2.7 kb in length, 1.0 kb, 500 bp, 250 bp or 100 bp in length. The enhancer may also include a plurality of copies of SEQ ID NO:23 or SEQ ID NO:20.
The enhancer including SEQ ID NO:20 or SEQ ID NO:23 may be immediately contiguous to a polypeptide-encoding DNA. Alternatively, the enhancer may be separated by 5, 10, 20, 30, 40, 50, 75, or 100 nucleotides from the polypeptide-encoding DNA. In addition to or alternatively, the enhancer may be contiguous to, or be separated by 5, 10, 20, 30, 40, 50, 75, or 100 nucleotides from an APEG-1 promoter or a heterologous promoter.
Preferably, expression of a polypeptide under the control of the APEG enhancer (e.g., SEQ ID No:17, SEQ ID NO:20, or SEQ ID NO:23) is at least 50% greater (e.g., as measured in the amount of polypeptide-encoding mRNA transcript), preferably at least 100% greater, more preferably at least 200% greater, and still more preferably at least 400% greater in vascular smooth muscle cells than in non-vascular smooth muscle cells. Most preferably, the APEG-1 enhancer directs vascular smooth muscle cell-specific polypeptide expression and directs negligible polypeptide expression in non-smooth muscle cell types. The enhancer sequence may in addition regulate developmental stage-specific expression, e.g., preferential expression in embryonic cells, of a polypeptide-encoding sequence.
The DNA of the invention (enhancer sequence) may be operably linked to a DNA sequence encoding a polypeptide that is not APEG-1 (i.e., a heterologous polypeptide), and function to regulate vascular smooth muscle cell-specific transcription of the polypeptide-encoding sequence. Examples of such polypeptides include tissue plasminogen activator (tPA), p21 cell cycle inhibitor, nitric oxide synthase, interferon-xcex3, and atrial natriuretic polypeptide.
The invention also includes a vector containing the enhancer DNA of the invention (operably linked to a polypeptide-encoding DNA sequence) and a vascular smooth muscle cell containing the vector. Also within the invention is a method of directing vascular smooth cell-specific expression of the polypeptide by introducing the vector into a vascular smooth muscle cell and maintaining the cell under conditions which permit expression of the polypeptide, e.g., introducing the vector into a human patient for gene therapy.
The vector of the invention can be used for gene therapy. For example, the vector can be introduced into a vascular smooth muscle cell to direct vascular smooth muscle cell-specific expression of a polypeptide. The vector of the invention can also be used for directing developmental stage-specific expression, e.g., preferential expression by embryonic cells, of a polypeptide, involving introducing into a vascular smooth muscle cell the vector of the invention.
The invention also features a method of inhibiting proliferation of vascular smooth muscle cells by administering to the cells an APEG-1 polypeptide.
The invention also features a striated muscle cell-specific variant gene product arising from the same genomic DNA encoding APEG-1, and therefore provides a substantially pure DNA (e.g., genomic DNA, cDNA or synthetic DNA) encoding a striated muscle preferentially-expressed gene (SPEG) polypeptide.
The DNA may encode a naturally occurring mammalian SPEG polypeptide such as a human SPEG polypeptide (SEQ ID NO:14) or mouse SPEG polypeptide (SEQ ID NO:16). For example, the invention includes degenerate variants of the human cDNA (SEQ ID NO:13) or the mouse cDNA (SEQ ID NO:15). The invention also includes a substantially pure DNA comprising a strand which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO:13 or 15, or the complements thereof.
A substantially pure DNA having at least 50% sequence identity (preferably at least 70%, more preferably at least 80%, and most preferably at least 90%) to SEQ ID NO:13, or 15, and encoding a polypeptide having a biological activity of a SPEG polypeptide is also within the invention.
The invention also includes a substantially pure DNA containing a constitutive or inducible striated muscle cell-specific promoter, e.g., a SPEG promoter which is preferably in a vector into which an heterologous gene may be or has been cloned, and under the control of which promoter the gene may be expressed. The promoter is preferably specific for striated muscle cells (e.g., cells of skeletal or cardiac muscle). DNA encoding SPEG may be operably linked to such regulatory sequences for expression of the SPEG polypeptide in striated muscle cells.
The invention also provides a method of directing striated muscle cell-specific expression of a protein by introducing into a cell an isolated DNA containing a sequence encoding the protein operably linked to the striated cell-specific promoter. A cell containing the DNA or vector of the invention is also within the invention.
The invention also features a substantially pure SPEG polypeptide (e.g., human (SEQ ID NO:14) or mouse SPEG (SEQ ID NO:16) and an antibody which specifically binds to a SPEG polypeptide.
The invention further features a substantially pure DNA having an APEG-1 derived cis-acting transcriptional repressor sequence. A xe2x80x9ccis-acting transcriptional repressorxe2x80x9d as used herein is a nucleic acid which functions to decrease transcription of an operably linked nucleic acid sequence. For example, transcription of an operably linked sequence is decreased when a trans-acting repressor, e.g., an endogenous intracellular protein, binds to the cis-acting repressor sequence. Inhibiting binding of the trans-acting repressor, e.g., by administering an exogenous compound that binds to the cis-acting transcriptional repressor, leads to derepression and a concomitant increase in expression of the operably-linked nucleic acid.
The cis-acting transcriptional repressor sequence may be linked to other cis-acting elements, e.g., additional copies of the cis-acting transcriptional repressor sequence. The elements may be contiguous or separated by DNA not involved in the regulation of transcription, e.g., a transcriptional repressor element may be in a position immediately adjacent to the promoter element or up to several kilobases upstream or downstream of the transcriptional start site.
In some embodiments, the cis-acting transcriptional repressor sequence hybridizes to a sequence including nucleotides xe2x88x923337 to xe2x88x922663 of the 5xe2x80x2 region of the mouse APEG-1 gene (SEQ ID NO:24), or its complement. In other embodiments, the cis-acting transcriptional repressor sequence comprises SEQ ID NO:24, or its complement.
The cis-acting transcriptional repressor sequence can be, e.g., less than 4.0 kb, 3.0 kb, 1.5 kb, 1.0 kb, or even 670 nucleotides in length. In some embodiments, the nucleic acid continuing the cis-acting transcriptional repressor sequence does not include the sequence of SEQ ID NO:23.
The invention also includes a vector comprising the cis-acting transcriptional repressor sequence.
In a further aspect, the invention includes a method of evaluating a compound for the ability to bind to a vascular smooth muscle cell cis-acting transcriptional repressor sequence. The method includes contacting the compound with a vascular smooth muscle cell cis-acting transcriptional repressor sequence and determining the amount of binding of the compound to the vascular smooth cell cis-acting transcriptional repressor sequence. The cis-acting transcriptional repressor sequence can be, for example, a sequence which hybridizes under high stringency conditions to SEQ ID NO:24 and can be SEQ ID NO:24.
In a further aspect, the invention includes a method of evaluating a compound for the ability to bind to a cis-acting transcriptional repressor sequence. In this method, the compound is contacted with a vascular smooth muscle cell containing a nucleic acid comprising a vascular smooth muscle cell cis-acting transcriptional repressor sequence operably linked to a sequence encoding a reporter molecule is contacted. The amount of the reporter molecule expressed by the cell is measured, and an alteration in the level of reporter molecule expressed in the presence of the compound compared to the level in the absence of the compound indicates that the compound binds to a vascular smooth muscle cell cis-acting transcriptional repressor sequence.
In another aspect, the invention includes a method of evaluating a compound for the ability to bind to increase APEG-1 expression. In this method, a vascular smooth muscle cell is contacted with a test compound, and the amount of APEG-1 transcript or polypeptide in the vascular smooth muscle cell is measured. An increase in the amount of APEG-1 transcript or gene product indicates the compound increases APEG-1 expression.
In a further aspect, the invention includes a method of inhibiting vascular smooth muscle cell proliferation by contacting a compound which binds to a vascular smooth muscle cell cis-acting transcriptional repressor sequence. The compound is provided in an amount effective to inhibit proliferation of the vascular smooth muscle cell. In some embodiments, the cis-acting transcriptional repressor sequence includes a sequence which hybridizes at high stringency to SEQ ID NO:24. For example, the cis-acting transcriptional repressor sequence comprises SEQ ID NO:24.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.